Biological fermentation feed and preparation method thereofTechnical field
The present invention relates to biological fermentation feed and preparation method thereof, belong to solid feed field.
Background technology
Along with the raising of people's living standard, the demand for pollution-free food, pollution-free food is more and more higher, and pork and internal organs thereof receive much concern especially as the meat that Chinese people are traditional.At present, the common feedstuffs kind of pig is a lot, is divided into the large class of protein feeds, energy feed, roughage, green forage, ensilage, mineral feed and feed addictive 8 by nutrition.Although all kinds of feed has respective distinctive effect, also have requirement to the feed collocation of the pig of various different cultivars, the ANFs particularly in vegetalitas, animality and mineral material is unfavorable for digesting and assimilating of pig.Meanwhile, in the production of pork pig, cause excessive use antibiotic and abuse prohibited items because of eager for instant success and quick profits, the harmful substance in animal food is residual may exceed standard, and has a strong impact on the product quality of pork, thus affects the healthy of people.Therefore, for the different approaches of pork product, mode and degree, research environment-friendly feed substitute, replaces those feed being easy to cause pork to pollute or additives, thus produce the pork product of high-quality safety-type, become a new trend of future in feed industry.
Biological fermentation feed refers under manual control condition, microorganism is by the metabolic activity of self, decomposed by ANFs in vegetalitas, animality and mineral material or transform, generation more can be searched for food by livestock and poultry, digests, absorb and the feedstuff of nonhazardous effect.Show according to the study, pig is with after the complete feed effect of undergoing microbial fermentation, and produce a large amount of lactic acid bacterias in feed, acetic acid and lactic acid concn are greatly enhanced, significantly reduce the pH value of feed, fermented feed of feeding can improve the growth performance of pig and improve the intestinal health of pig.Current biological fermentation feed exists that bacterial classification source is complicated, trophic level is low, to defects such as the enteron aisle improvement result of pig are limited, these are not only relevant to fermented bacterium and zymotechnique, and fermentation substrate also has a certain impact to it.
Patent of invention CN102499321A discloses a kind of Nonreactive fermented feed of mixed bacteria and preparation method thereof.This feed is mixed with the butt matter of mixed feed by the bacterium liquid of compound bacteria, is fermented, the premix of rear interpolation 4% of having fermented and making, wherein, compound bacteria by bacillus subtilis, bite Lactobacillus lactis and S. cervisiae forms, proportioning is 1:1:1, and butt matter is made up of the component of following percentage by weight: dregs of beans 51.81%, corn protein powder 22.02%, rapeseed dregs 3.89%, peanut meal 3.89%, calcium monohydrogen phosphate 5.18%, salt 2.98%, lysine 1.68%, Choline Chloride 0.13%.This feed does not add any antibiotic, formed by three kinds of probiotics fermentions, growth of animal can be promoted, and maintenance gut flora balance is had certain effect, but the butt matter cost of this feed is higher, and also need after the fermentation was completed to add the premix be made up of vitamin, biotin, Choline Chloride etc., complex manufacturing, cost is higher.
Normally, common probio obtains as bacillus subtilis, candidiasis, British mold, Bacillus acidi lactici, Bifidobacterium all can be separated from the ight soil of animal intestinal and humans and animals, Bifidobacterium (separation of Bifidobacterium, Isolation and characterization is obtained as being separated in infant faeces, old man's ight soil, honeybee enteron aisle with people such as Wen Xiang, northwest agricultural journal, 1999,8 (4): 97-101); Liu Yu is separated and obtains Bacillus acidi lactici (Isolation and ldentification of surrounding area, Qiqihar intestine of young pigs Bacillus acidi lactici, pig industry science, the 7th phase in 2011,94-95) in chitling road; The people such as Zhou Honglu are separated and obtain bacillus subtilis (the separation qualification of a bacillus subtilis and prebiotic potential quality analysis thereof, China brewages, the 12nd phase in 2010,137-139) in swine excrement; Sunshine gorgeous grade in field is separated multiple saccharomycete such as obtaining British mold, pityrosporum ovale bacterium, candidiasis (the saccharomycetic separation in chitling road and Preliminary Identification, feed is studied, the 10th phase in 2011,42-43) in chitling road.
Summary of the invention
Technical problem to be solved by this invention is to provide biological fermentation feed and preparation method thereof, and biological fermentation feed of the present invention containing antibiotic, not only can not promote growth of animal, can also improve and maintain intestinal microflora and stablize.
Biological fermentation feed of the present invention, fermented by composite bacteria liquid and fermentation substrate to form, wherein, composite bacteria liquid is by bacillus subtilis, mulriple yeasts, Bacillus acidi lactici and Bifidobacterium composition, mulriple yeasts is made up of candidiasis and British mold, fermentation substrate is made up of the component of following weight portion: corn 30 ~ 35 weight portion, dregs of beans 10 ~ 15 weight portion, wash rice bran 25 ~ 35 weight portion, wheat bran 10 ~ 15 weight portion, dusty yeast 3 ~ 8 weight portion, sodium chloride 0.5 ~ 2 weight portion, calcium monohydrogen phosphate 1 ~ 5 weight portion, soybean oil 0 ~ 3 weight portion, manganese sulfate 0.5 ~ 1.5 weight portion, magnesium sulfate 0.5 ~ 1.5 weight portion.
In the mulriple yeasts of biological fermentation feed of the present invention, by number ratio, candidiasis: British mold=1:1 ~ 3; In composite bacteria liquid, bacillus subtilis: mulriple yeasts: Bacillus acidi lactici: Bifidobacterium=190 ~ 210:1:7 ~ 12:7 ~ 12, preferred candidiasis: British mold=1:2; Preferred bacillus subtilis: mulriple yeasts: Bacillus acidi lactici: Bifidobacterium=200:1:10:10.Namely four kinds of bacteriums in composite bacteria liquid of the present invention can carry out Liquid Culture respectively, get respective volume fluid nutrient medium during use to mix, its volume ratio is preferably bacillus subtilis: mulriple yeasts: Bacillus acidi lactici: Bifidobacterium=2:1:1:1, and wherein, bacillus subtilis bacteria concentration is with 109cFU/ml counts; Mulriple yeasts is with 107cFU/ml counts; Bacillus acidi lactici concentration is with 108cFU/ml counts; Bifidobacterium concentration is with 108cFU/ml counts.
Wherein, in order to improve feed efficiency, fermentation substrate is preferably made up of the component of following weight portion: corn 32 weight portion, dregs of beans 13 weight portion, wash rice bran 30 weight portion, wheat bran 12 weight portion, dusty yeast 5 weight portion, sodium chloride 1 weight portion, calcium monohydrogen phosphate 3 weight portion, soybean oil 2 weight portion, manganese sulfate 1 weight portion, magnesium sulfate 1 weight portion.
Further, bacillus subtilis in biological fermentation feed composite bacteria liquid of the present invention, candidiasis, British mold, Bacillus acidi lactici and Bifidobacterium obtain by being separated in health pig body, extracting, its field planting enteron aisle and to maintain the stable ability of intestinal microflora stronger again of the feed obtained by such strain fermentation.
Further, the inoculum concentration of composite bacteria liquid of the present invention in fermentation substrate is 0.08 ~ 0.12ml/g, in 100g fermentation substrate, namely add the composite bacteria liquid of 8 ~ 12ml, wherein, composite bacteria liquid with the concentration of bacillus for 109cFU/ml counts.The inoculum concentration of preferred composite bacteria liquid in fermentation substrate is 0.1ml/g.
Further, the fermentation condition of biological fermentation feed of the present invention is as follows: the water content of fermentation substrate is 60 ~ 65%, and fermentation time is 5 ~ 7 days, and fermentation temperature is not less than 20 DEG C, and the water content of preferred fermentation substrate is 65%.
The present invention also provides the preparation method of biological fermentation feed of the present invention, and its step is as follows:
A, be separated in health pig body, extract bacillus subtilis, mulriple yeasts, Bacillus acidi lactici and Bifidobacterium, and to preserve;
B, by bacillus subtilis, mulriple yeasts, Bacillus acidi lactici and Bifidobacterium mixing, and to be inoculated in the fluid nutrient medium that is made up of 15% molasses and 5% whey powder by the inoculum concentration of 3%, to ferment 2 ~ 3 days;
C, be inoculated on fermentation substrate by the bacterium liquid fermented, regulate its moisture, sealing and fermenting 5 ~ 7 days, obtains biological fermentation feed.
Wherein, the isolation and identification method of bacillus subtilis, mulriple yeasts, Bacillus acidi lactici and Bifidobacterium can adopt the method for this area routine, and following methods also can be adopted to carry out:
The separation and Extraction of bacillus subtilis: get healthy adult pig fresh excreta 4g in 50ml sterile saline, 180r/min80 DEG C of vibration 15min; Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on nutrient agar panel, under 37 DEG C of conditions, cultivate 48h; Picking colony is white or milky, and centre has the starlike single bacterium colony pursed up to carry out 2 ~ 3 purifying cultivations again; Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores; The qualification of bacillus subtilis: a little above-mentioned preservation bacterium of picking, carries out Starch Hydrolysis test, gelatin liquefaction test, product Protease assays, nitrate reduction test, indole test, bacteriostatic test respectively; If above-mentioned result of the test only has indole test to be negative, other is all positive, and in bacteriostatic test, inhibition zone is greater than 14mm, then judge that gained bacterium is as required bacillus subtilis.
The separation and Extraction of mulriple yeasts: choose the healthy adult pig after slaughtering, choose jejunum and cut off, gets Digesta samples and loads in sealed bag, 4 DEG C of Refrigerator stores; Get 1g sample, add physiological saline 100ml, shaking flask 15min in the constant-temperature table of 180r/min and 37 DEG C; Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on wort agar flat board, cultivate 48h under 37 DEG C of conditions; Picking colony is white or milky, has single bacterium colony of mycelia to carry out 2 ~ 3 purifying again and cultivates; Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores; The qualification of mulriple yeasts: a little above-mentioned preservation bacterium of picking, carries out Starch Hydrolysis test, nitrate reduction test, ethanol assimilation experiments, glucose sugar fermentating test, bacteriostatic test respectively; If above-mentioned test is all positive, and in bacteriostatic test, inhibition zone is greater than 14mm, then judge that gained bacterium is as required mulriple yeasts.
The separation and Extraction of lactic acid bacteria: get sodium selenite fresh excreta 4g in 50ml sterile saline, 180r/min vibrates 5min; Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on MRS flat board, Anaerobic culturel 48h under 37 DEG C of conditions; Picking colony is white or canescence, and the coarse single bacterium colony of circular surface carries out 2 ~ 3 purifying again and cultivates; Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores; The qualification of lactic acid bacteria: a little above-mentioned preservation bacterium of picking, carries out experiment of growth factor, nitrate reduction test, mobility test, catalase test, gelatin liquefaction test, indole test respectively; Above-mentioned result of the test only has experiment of growth factor and bacteriostatic test for positive, and in bacteriostatic test, inhibition zone is greater than 14mm, and other is feminine gender, then judge that gained bacterium is as required lactic acid bacteria.
The separation and Extraction of Bifidobacterium: get sodium selenite fresh excreta 4g in 50ml sterile saline, 180r/min vibrates 5min; Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on BL flat board, Anaerobic culturel 48h under 37 DEG C of conditions; Picking colony is that milky single bacterium colony carries out 2 ~ 3 purifying cultivations again; Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores; The qualification of Bifidobacterium: a little above-mentioned preservation bacterium of picking, carries out experiment of growth factor, nitrate reduction test, catalase test, gelatin liquefaction test, indole test respectively; If the above results only has experiment of growth factor and bacteriostatic test for positive, and in bacteriostatic test, inhibition zone is greater than 14mm, other is feminine gender, then judge that gained bacterium is as required Bifidobacterium.
Biological fermentation feed of the present invention can be used as feed addictive and uses, and half an hour before feeding, biological fermentation feed of the present invention and common pig feed are mixed, and with water, feed surface is soaked and can be used, its addition can be determined by raising demand.General, piglet addition of feeding is 20%, and child care pig addition is 25%, and growing and fattening pigs and kind, boar addition are 30%.
Compared with prior art, biological fermentation feed of the present invention has the following advantages:
1, biological fermentation feed antibiotic-free of the present invention, green safety, can be added in daily feed in a large number, can guarantee the useful number of viable entered in pig body, can reduce the incidence of disease of various diseases, particularly can reduce the incidence of disease of disease of digestive tract.
2, the zymogenic growth and breeding of biological fermentation feed primary biological of the present invention, be hydrolyzed all kinds of nutriment, thus obtain the trophic factors that monose, each amino acid, aliphatic acid etc. can directly absorb, reduce digestion power consumption, therefore raw material sources is extensive, operability is large, benefit is obvious.
3, biological fermentation feed of the present invention is with low cost, has strong wine flavour, can improve appetite of entirely mining massively, and after using, each stage pig feed intake can improve 12%, and pig feed cost can reduce by 13%.
4, the composite bacteria liquid of biological fermentation feed of the present invention be separated in healthy big porker enteron aisle, the beneficial bacterium that extracts, it is field planting enteron aisle and to maintain the biofermentation bacterium that the stable ability of intestinal microflora obtains compared with other approach strong again; And suppress the pathogenic bacteria such as Escherichia coli, salmonella ability comparatively strong, there is obvious control action to full group's disease of digestive tract; Fermented bacterium is nontoxic, safety, the bacterial strain useful to biological growth and development.
Detailed description of the invention
Biological fermentation feed of the present invention, fermented by composite bacteria liquid and fermentation substrate to form, wherein, composite bacteria liquid is by bacillus subtilis, mulriple yeasts, Bacillus acidi lactici and Bifidobacterium composition, mulriple yeasts is made up of candidiasis and British mold, fermentation substrate is made up of the component of following weight portion: corn 30 ~ 35 weight portion, dregs of beans 10 ~ 15 weight portion, wash rice bran 25 ~ 35 weight portion, wheat bran 10 ~ 15 weight portion, dusty yeast 3 ~ 8 weight portion, sodium chloride 0.5 ~ 2 weight portion, calcium monohydrogen phosphate 1 ~ 5 weight portion, soybean oil 0 ~ 3 weight portion, manganese sulfate 0.5 ~ 1.5 weight portion, magnesium sulfate 0.5 ~ 1.5 weight portion.
In the mulriple yeasts of biological fermentation feed of the present invention, by number ratio, candidiasis: British mold=1:1 ~ 3; In composite bacteria liquid, bacillus subtilis: mulriple yeasts: Bacillus acidi lactici: Bifidobacterium=190 ~ 210:1:7 ~ 12:7 ~ 12, preferred candidiasis: British mold=1:2; Preferred bacillus subtilis: mulriple yeasts: Bacillus acidi lactici: Bifidobacterium=200:1:10:10.Namely four kinds of bacteriums in composite bacteria liquid of the present invention can carry out Liquid Culture respectively, get respective volume fluid nutrient medium during use to mix, its volume ratio is preferably bacillus subtilis: mulriple yeasts: Bacillus acidi lactici: Bifidobacterium=2:1:1:1, and wherein, bacillus subtilis bacteria concentration is with 109cFU/ml counts; Mulriple yeasts is with 107cFU/ml counts; Bacillus acidi lactici concentration is with 108cFU/ml counts; Bifidobacterium concentration is with 108cFU/ml counts.
Wherein, in order to improve feed efficiency, fermentation substrate is preferably made up of the component of following weight portion: corn 32 weight portion, dregs of beans 13 weight portion, wash rice bran 30 weight portion, wheat bran 12 weight portion, dusty yeast 5 weight portion, sodium chloride 1 weight portion, calcium monohydrogen phosphate 3 weight portion, soybean oil 2 weight portion, manganese sulfate 1 weight portion, magnesium sulfate 1 weight portion.
Further, bacillus subtilis in biological fermentation feed composite bacteria liquid of the present invention, candidiasis, British mold, Bacillus acidi lactici and Bifidobacterium obtain by being separated in health pig body, extracting, its field planting enteron aisle and to maintain the stable ability of intestinal microflora stronger again of the feed obtained by such strain fermentation.
Further, the inoculum concentration of composite bacteria liquid of the present invention in fermentation substrate is 0.08 ~ 0.12ml/g, in 100g fermentation substrate, namely add the composite bacteria liquid of 8 ~ 12ml, wherein, composite bacteria liquid with the concentration of bacillus for 109cFU/ml counts.The inoculum concentration of preferred composite bacteria liquid in fermentation substrate is 0.1ml/g.
Further, the fermentation condition of biological fermentation feed of the present invention is as follows: the water content of fermentation substrate is 60 ~ 65%, and fermentation time is 5 ~ 7 days, and fermentation temperature is not less than 20 DEG C, and the water content of preferred fermentation substrate is 65%.
The present invention also provides the preparation method of biological fermentation feed of the present invention, and its step is as follows:
A, be separated in health pig body, extract bacillus subtilis, mulriple yeasts, Bacillus acidi lactici and Bifidobacterium, and to preserve;
B, by bacillus subtilis, mulriple yeasts, Bacillus acidi lactici and Bifidobacterium mixing, and to be inoculated in the fluid nutrient medium that is made up of 15% molasses and 5% whey powder by the inoculum concentration of 3%, to ferment 2 ~ 3 days;
C, be inoculated on fermentation substrate by the bacterium liquid fermented, regulate its moisture, sealing and fermenting 5 ~ 7 days, obtains biological fermentation feed.
Wherein, the isolation and identification method of bacillus subtilis, mulriple yeasts, Bacillus acidi lactici and Bifidobacterium can adopt the method for this area routine, and following methods also can be adopted to carry out:
The separation and Extraction of bacillus subtilis: get healthy adult pig fresh excreta 4g in 50ml sterile saline, 180r/min80 DEG C of vibration 15min; Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on nutrient agar panel, under 37 DEG C of conditions, cultivate 48h; Picking colony is white or milky, and centre has the starlike single bacterium colony pursed up to carry out 2 ~ 3 purifying cultivations again; Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores; The qualification of bacillus subtilis: a little above-mentioned preservation bacterium of picking, carries out Starch Hydrolysis test, gelatin liquefaction test, product Protease assays, nitrate reduction test, indole test, bacteriostatic test respectively; If above-mentioned result of the test only has indole test to be negative, other is all positive, and in bacteriostatic test, inhibition zone is greater than 14mm, then judge that gained bacterium is as required bacillus subtilis.
The separation and Extraction of mulriple yeasts: choose the healthy adult pig after slaughtering, choose jejunum and cut off, gets Digesta samples and loads in sealed bag, 4 DEG C of Refrigerator stores; Get 1g sample, add physiological saline 100ml, shaking flask 15min in the constant-temperature table of 180r/min and 37 DEG C; Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on wort agar flat board, cultivate 48h under 37 DEG C of conditions; Picking colony is white or milky, has single bacterium colony of mycelia to carry out 2 ~ 3 purifying again and cultivates; Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores; The qualification of mulriple yeasts: a little above-mentioned preservation bacterium of picking, carries out Starch Hydrolysis test, nitrate reduction test, ethanol assimilation experiments, glucose sugar fermentating test, bacteriostatic test respectively; If above-mentioned test is all positive, and in bacteriostatic test, inhibition zone is greater than 14mm, then judge that gained bacterium is as required mulriple yeasts.
The separation and Extraction of lactic acid bacteria: get sodium selenite fresh excreta 4g in 50ml sterile saline, 180r/min vibrates 5min; Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on MRS flat board, Anaerobic culturel 48h under 37 DEG C of conditions; Picking colony is white or canescence, and the coarse single bacterium colony of circular surface carries out 2 ~ 3 purifying again and cultivates; Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores; The qualification of lactic acid bacteria: a little above-mentioned preservation bacterium of picking, carries out experiment of growth factor, nitrate reduction test, mobility test, catalase test, gelatin liquefaction test, indole test respectively; Above-mentioned result of the test only has experiment of growth factor and bacteriostatic test for positive, and in bacteriostatic test, inhibition zone is greater than 14mm, and other is feminine gender, then judge that gained bacterium is as required lactic acid bacteria.
The separation and Extraction of Bifidobacterium: get sodium selenite fresh excreta 4g in 50ml sterile saline, 180r/min vibrates 5min; Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on BL flat board, Anaerobic culturel 48h under 37 DEG C of conditions; Picking colony is that milky single bacterium colony carries out 2 ~ 3 purifying cultivations again; Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores; The qualification of Bifidobacterium: a little above-mentioned preservation bacterium of picking, carries out experiment of growth factor, nitrate reduction test, catalase test, gelatin liquefaction test, indole test respectively; If the above results only has experiment of growth factor and bacteriostatic test for positive, and in bacteriostatic test, inhibition zone is greater than 14mm, other is feminine gender, then judge that gained bacterium is as required Bifidobacterium.
Biological fermentation feed of the present invention can be used as feed addictive and uses, and half an hour before feeding, biological fermentation feed of the present invention and common pig feed are mixed, and with water, feed surface is soaked and can be used, its addition can be determined by raising demand.General, piglet addition of feeding is 20%, and child care pig addition is 25%, and growing and fattening pigs and kind, boar addition are 30%.
Below in conjunction with embodiment, the specific embodiment of the present invention is further described, does not therefore limit the present invention among described scope of embodiments.
Embodiment 1
1, the preparation of fermentation substrate
Get corn 30kg, dregs of beans 10kg, wash rice bran 25kg, wheat bran 10kg, dusty yeast 3kg, sodium chloride 0.5kg, calcium monohydrogen phosphate 1kg, manganese sulfate 0.5kg, magnesium sulfate 0.5kg, mixing, obtains fermentation substrate A.
Get corn 35kg, dregs of beans 15kg, wash rice bran 35kg, wheat bran 15kg, dusty yeast 8kg, sodium chloride 2kg, calcium monohydrogen phosphate 5kg, soybean oil 3kg, manganese sulfate 1.8kg, magnesium sulfate 1.8kg, mixing, obtains fermentation substrate B.
Get corn 32kg, dregs of beans 13kg, wash rice bran 30kg, wheat bran 12kg, dusty yeast 5kg, sodium chloride 1kg, calcium monohydrogen phosphate 3kg, soybean oil 2kg, manganese sulfate 1kg, magnesium sulfate 1kg, mixing, obtains fermentation substrate C.
2, the separation qualification of bacterial classification
2.1, the separation and Extraction qualification of bacillus subtilis
A. the separation and Extraction of bacillus subtilis:
Get healthy adult pig fresh excreta 4g in 50ml sterile saline, 180r/min80 DEG C of vibration 15min; (when without specified otherwise, in the embodiment of the present invention, pig used and pig fresh excreta are all from Miyi County ten thousand people agriculture and animal husbandry Co., Ltd pig farm.)
Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on nutrient agar panel, under 37 DEG C of conditions, cultivate 48h; Picking colony is white or milky, and centre has the starlike single bacterium colony pursed up to carry out 2 ~ 3 purifying cultivations again;
Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores;
B. the qualification of bacillus subtilis:
The a little above-mentioned preservation bacterium of picking, carries out Starch Hydrolysis test, gelatin liquefaction test, product Protease assays, nitrate reduction test, indole test, bacteriostatic test respectively;
If above-mentioned result of the test only has indole test to be negative, other is all positive, and in bacteriostatic test, inhibition zone is greater than 14mm, then judge that gained bacterium is as required bacillus subtilis.
2.2, the separation and Extraction qualification of mulriple yeasts
A. the separation and Extraction of mulriple yeasts:
Choose the healthy adult pig after slaughtering, choose jejunum and cut off, get Digesta samples and load in sealed bag, 4 DEG C of Refrigerator stores;
Get 1g sample, add physiological saline 100ml, shaking flask 15min in the constant-temperature table of 180r/min and 37 DEG C;
Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on wort agar flat board, cultivate 48h under 37 DEG C of conditions; Picking colony is white or milky, has single bacterium colony of mycelia to carry out 2 ~ 3 purifying again and cultivates;
Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores;
B. the qualification of mulriple yeasts:
The a little above-mentioned preservation bacterium of picking, carries out Starch Hydrolysis test, nitrate reduction test, ethanol assimilation experiments, glucose sugar fermentating test, bacteriostatic test respectively;
If above-mentioned test is all positive, and in bacteriostatic test, inhibition zone is greater than 14mm, then judge that gained bacterium is as required mulriple yeasts.
2.3, the separation and Extraction qualification of lactic acid bacteria
A. the separation and Extraction of lactic acid bacteria:
Get sodium selenite fresh excreta 4g in 50ml sterile saline, 180r/min vibrates 5min;
Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on MRS flat board, Anaerobic culturel 48h under 37 DEG C of conditions; Picking colony is white or canescence, and the coarse single bacterium colony of circular surface carries out 2 ~ 3 purifying again and cultivates;
Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores;
B. the qualification of lactic acid bacteria:
The a little above-mentioned preservation bacterium of picking, carries out experiment of growth factor, nitrate reduction test, mobility test, catalase test, gelatin liquefaction test, indole test respectively;
If above-mentioned result of the test only has experiment of growth factor and bacteriostatic test for positive, and in bacteriostatic test, inhibition zone is greater than 14mm, other is feminine gender, then judge that gained bacterium is as required lactic acid bacteria.
2.4, the separation and Extraction authentication method of Bifidobacterium
A. the separation and Extraction of Bifidobacterium:
Get sodium selenite fresh excreta 4g in 50ml sterile saline, 180r/min vibrates 5min;
Get the sample gradient handled well and be diluted to 10-9, select 10-7, 10-8, 10-9three gradients are coated on BL flat board, Anaerobic culturel 48h under 37 DEG C of conditions; Picking colony is that milky single bacterium colony carries out 2 ~ 3 purifying cultivations again;
Picking a little purifying gained bacterium, microscope inspection; And 4 DEG C of Refrigerator stores;
B. the qualification of Bifidobacterium:
The a little above-mentioned preservation bacterium of picking, carries out experiment of growth factor, nitrate reduction test, catalase test, gelatin liquefaction test, indole test respectively;
If the above results only has experiment of growth factor and bacteriostatic test for positive, and in bacteriostatic test, inhibition zone is greater than 14mm, other is feminine gender, then judge that gained bacterium is as required Bifidobacterium.
3, the preparation of composite bacteria liquid
The four kinds of bacteriums above-mentioned screening obtained carry out Liquid Culture respectively, and get the mixing of respective volume fluid nutrient medium during use, its volume ratio is bacillus subtilis: mulriple yeasts: lactic acid bacteria: Bifidobacterium=2:1:1:1, and now, bacillus subtilis bacteria concentration is with 109cFU/ml counts; Mulriple yeasts is with 107cFU/ml counts; Lactic acid bacteria concentration is with 108cFU/ml counts; Bifidobacterium concentration is with 108cFU/ml counts.
By the bacterium liquid mixed by 3% inoculum concentration be inoculated in the fluid nutrient medium that is made up of 15% molasses and 5% whey powder, ferment 2 ~ 3 days, now, fluid nutrient medium gives out strong sour fragrance, and wherein, the concentration of Bifidobacterium is 109cFU/ml.
4, the preparation of biological fermentation feed
The composite bacteria liquid of above-mentioned preparation is inoculated on fermentation substrate, and adds appropriate cold boiling water and regulate its moisture about 65%, now, firmly to hold with a firm grip a fermentation substrate, fist is sewed with watermark but without dripping, is dispensed in sealing bag and carries out fermenting to giving out strong wine flavour, obtaining biological fermentation feed.Wherein, inoculum concentration and fermentation condition are in table 1.
Table 1
| Biological fermentation feed is numbered | Fermentation substrate | Inoculum concentration | Fermentation time | Fermentation temperature (DEG C) |
| 1 | Fermentation substrate A | 8％ | 5 days | 20 |
| 2 | Fermentation substrate B | 11％ | 6 days | 25 |
| 3 | Fermentation substrate C | 12％ | 7 days | 28 |
| 4 | Fermentation substrate C | 10％ | 5 days | 21 |
Test example 1
The biological fermentation feed 1 ~ 4 of embodiment 1 gained is applied to during piglet feeds.Experimental group 1 ~ 4 is respectively to add biological fermentation feed 1 ~ 4 (namely experimental group 1 is add the biological fermentation feed 1 in embodiment 1 in daily feed, experimental group 2 is add the biological fermentation feed 2 in embodiment 1, experimental group 3 is add the biological fermentation feed 3 in embodiment 1, experimental group 4 is add the biological fermentation feed 4 in embodiment 1), its addition accounts for 20% of total forage volume.If two groups of contrasts, control group 1 is daily feed; Control group 2 adds following biological fermentation feed in daily feed: the composite bacteria liquid of employing is that commercially available bacterial classification configuration forms (bacillus subtilis, candidiasis, British mold, Bacillus acidi lactici and Bifidobacterium are commercially available), its fermentation condition is identical with the condition of biological fermentation feed 4, and its addition accounts for 20% of total forage volume.Often organize 30 sucking pigs, repeat for ten times, average, statistics is in table 2.
Table 2
| Experimental group | Diarrhea rate (%) | Wean sucking pig average weight (Kg) |
| Experimental group 1 | 17.7 | 7.0 |
| Experimental group 2 | 15.3 | 7.2 |
| Experimental group 3 | 16.3 | 7.0 |
| Experimental group 4 | 16.0 | 7.1 |
| Control group 1 | 35.7 | 6.5 |
| Control group 2 | 23.3 | 6.9 |
As can be seen from Table 2, after adding biological fermentation feed of the present invention, grice diarrhoea rate can reduce by 20%, weanling pig average weight improves 0.6kg, as can be seen from the contrast of experimental group and control group 1, adopt the beneficial bacterium being separated in health pig enteron aisle, extracting, the biofermentation bacterium obtained than other approach is strong; And suppress the pathogenic bacteria such as Escherichia coli, salmonella ability comparatively strong, there is obvious control action to full group's disease of digestive tract.
Test example 2
The biological fermentation feed 1 ~ 4 of embodiment 1 gained is applied in feeding of growing and fattening pigs.Its experimental group and control group same test example 1 is set.Often organize 30 growing and fattening pigs, original body mass is 30 ± 1kg.Find after feeding, after adding biological fermentation feed of the present invention, growing and fattening pigs feedstuff-meat ratio is reduced to 3.0:1 by 3.2:1, and marketing time shifts to an earlier date 10 days.Concrete condition refers to table 3.Wherein, marketing time refers to that pig body weight reaches the time of 110kg.
Table 3
| Experimental group | Feedstuff-meat ratio | Marketing time (my god) |
| Experimental group 1 | 3.1:1 | 181 |
| Experimental group 2 | 2.9:1 | 178 |
| Experimental group 3 | 3.1:1 | 182 |
| Experimental group 4 | 3.0:1 | 180 |
| Control group 1 | 3.2:1 | 190 |
| Control group 2 | 3.0:1 | 185 |